The results showed that the 5-HT-sufficient group mice had a significantly lower survival rate than the 5-HT-deficient group in CLP-induced sepsis and septic shock. The KO-CLP sepsis group received a lower clinical score than the WT-CLP sepsis group. Meanwhile, the body temperature of mice in the KO-CLP sepsis group was higher than that in the WT-CLP sepsis group and was much closer to the normal body temperature 24 hours after CLP. The tissue histopathology analysis revealed that 5-HT markedly exacerbated histological damages in the peritoneum, lung, liver, kidney, intestinal tissue, and heart in sepsis. Moreover, significant lower levels of TNF-α, IL-6, bacterial loads, MPO, and ROS were discovered in the KO-CLP sepsis group in contrast to the WT-CLP sepsis group. In conclusion, 5-HT drives mortality and exacerbates organ dysfunction by promoting serum cytokines and bacterial loads as well as facilitating oxidative stress in the process of sepsis.Sepsis is defined as a life-threatening organ dysfunction caused by a dysregulated host response to infection with a high mortality. 5-Hydroxytryptamine (5-HT) is an important regulatory factor in inflammation. The aim of this study is to investigate the role of 5-HT on cecal ligation and puncture- (CLP-) induced sepsis in the mouse model. CLP was performed on C57B/6 wild-type (WT) mice and  The overproduction of inflammatory mediators induces endothelial and epithelial injury, vascular leakage, edema, and vasodilatation. In addition, the oxidative stress and mitochondrial dysfunction also exert vital roles in the organ damages and MODS [Sepsis is defined as a life-threatening organ dysfunction caused by a dysregulated host response to infection. It is characterized by high mortality between 28% and 50% and limited therapeutic options –3. In manitially . TNF-α aand MODS . The devSerotonin, or 5-hydroxytryptamine (5-HT), is an important regulatory factor in the gastrointestinal (GI) tract and other organ systems in addition to its role as a brain neurotransmitter . The majIt has been verified that gut microbiota regulates the host 5-HT biosynthesis and bacterial translocation contributes to the sepsis –24. BesiThe study was conducted using male TPH1 knockout mice  and wild-type C57BL/6 mice  . The TPH1−/− mice were originated from C57BL/6 mice. The lack of peripheral 5-HT was the only difference between TPH1−/− mice and C57BL/6 mice. The characteristics of TPH1−/− mice had been described previously , 30. Fivn = 6 for the sham groups and n = 15 for the experimental groups) were randomly divided as described above and were monitored to assess mortality for 5 days in the CLP sepsis model and 3 days in the CLP septic shock model. The temperature and clinical score of each animal was measured as previously described [The mice were randomly allocated into the following groups: (1) WT sham group: a sham laparotomy operation was performed on the WT mice. (2) KO sham group: a sham laparotomy operation was performed on TPH1 knockout mice. (3) WT-CLP sepsis group: the cecal ligation and puncture (CLP) with a 21-gauge needle was performed on WT mice. (4) KO-CLP sepsis group: the CLP with a 21-gauge needle was performed on TPH1 knockout mice. (5) WT+PCPA-CLP sepsis group: wild-type mice were pretreated with p-chlorophenylalanine . (6) KOescribed .The mice were anesthetized with chloral hydrate . The abdomen was disinfected with iodine volts, and a 2 cm midline abdominal incision was conducted. The cecum was exposed, ligated below the ileocecal valve with a 3-0 silk, and then punctured with a 21-gauge needle for the sepsis model or an 18-gauge needle for the septic shock model. After the operation, the cecum was placed back to the original location. Saline (30 ml/kg) was used for anesthesia recovery in the incubator. The mice were returned to their cages until full recovery , 33. Onlμm thickness were obtained and stained with hematoxylin and eosin (H&E). Then, two researchers assessed the histologic changes in a double-blinded way to select a representative field for future use through the light microscope.Six hours and 24 h after the CLP and sham operations, respectively, tissue samples from the peritoneum, lung, liver, kidney, intestines, and hearts were collected for histologic analysis. The tissues were fixed in 10% formalin for 48 hours and then treated with paraffin embedding. Serial sections of 5 At 6 h and 24 h after CLP, the mice were sacrificed to evaluate the acute peritonitis. Through a midline abdominal incision, 5 ml phosphate-buffered saline (PBS) was injected into the abdominal cavity. Then, all abdominal cavity liquid was collected to EP tubes. The exudate volume was calculated by subtracting the volume injected (5 ml) from the total volume. The mice serum was collected at 6 h and 24 h after the CLP. The liver and kidney injury was evaluated by measuring the level of serum alanine aminotransferase (ALT) and blood urea nitrogen (BUN). The activities of ALT and BUN were determined by an automated procedure in the Department of Inspection . Lung dysfunction was assessed by measuring the W/D weight ratio. The superior and middle lobes of the right lung were gathered for the assessment of the lung W/D ratio. The lung samples were weighed immediately after collection, then placed into an incubator at 80°C for 48 h to dry and weighed again. The lung W/D ratio was calculated by the wet weight divided by the dry weight. Intestine dysfunction was assessed by measuring the villus height in H&E staining. The heart dysfunction was assessed by the ejection fraction (echocardiography).α, IL-6, and IL-1β were measured with commercial ELISA kits according to the instructions of the manufacturer .The levels of serum TNF-At 24 h after the CLP, the mice were sacrificed. The peritoneal fluid, blood, and lung tissues were collected. After diluted with phosphate-buffered saline (PBS), the peritoneal fluid, blood, and mixed liquid of the lung tissues were cultured overnight on blood-agar base plates  at 37°C. The colony-forming units (CFUs) of peritoneal, blood, and lung bacterial loads were calculated.At 24 h after the CLP, the fresh lung and liver tissues of mice were harvested for tissue slides. The lung and liver tissue slides were fixed in 10% formaldehyde and blocked with 10% normal donkey serum diluted in PBS containing 0.1% triton. Then, the slides were incubated with a polyclonal rabbit anti-MPO antibody  diluted 1 : 200 in PBS for 1 hour at room temperature. The slides were washed and incubated with a donkey anti-rabbit antibody at 1 : 500 in PBS for 90 min at room temperature and counterstained with 4′6-diamidino-2-phenylindole (DAPI). The tissue sections were randomly screened (five fields/slide). Images were acquired using an inverted Leica CTR 6000 fluorescence microscope and merged with Leica Application Suite Advanced Fluorescence software .g for 15 min. The supernatants were obtained and stored at −80°C for the following analysis. The detection was conducted following the reference manual.The activity of MPO was measured using an activity assay kit (NanJing JianCheng Bioengineering). The liver and lung tissues were homogenized with PBS and centrifuged at 1500The fresh liver and lung tissues were collected and stored at −80°C. Five-millimeter thickness tissue sections were performed and incubated in Locke's buffer containing DHE concentration of 10 nM for 30 min. The lung tissue fluorescence  was observed by using fluorescence microscope.t-test was used for the comparison between the two groups. Kaplan-Meier curve was used for survival analysis and log-rank test for difference between the two groups. All tests were two-sided, and significance was accepted at p < 0.05. The statistical analyses were performed using GraphPad Prism software .The measurement data were expressed as mean ± SD. Student's p < 0.0001). Meanwhile, TPH1−/− mice treated with 5-HTP for 3 days could markedly replenish blood 5-HT in contrast to the TPH1−/− mice treated with saline .To validate the role of 5-HT in CLP-induced sepsis, the PCPA  and 5-HTP (the precursor of 5-HT) were injected to mice 3 days before CLP operation. The plasma 5-HT levels were measured to confirm the effects of PCPA and 5-HTP in the WT and TPH1−/− mice. The results showed that plasma 5-HT levels in the WT mice treated with PCPA for 3 days significantly decreased compared with that in the WT mice treated with saline  were randomly grouped and monitored to assess the 5-day survival rate in CLP-induced sepsis and the 3-day survival rate in CLP-induced septic shock (p = 0.0475). Furthermore, we conducted the experiment by adding PCPA and 5-HTP. The WT+PCPA-CLP sepsis group also showed an increased survival rate compared with the WT-CLP sepsis group (p = 0.045), while the survival rate of the KO+HTP-CLP sepsis group was lower than that of the KO-CLP sepsis group (p = 0.003). The mortality was revealed to be more serious in the CLP-induced septic shock model. Similar results were obtained for the septic shock model, demonstrating that the survival rate of the KO-CLP group was significantly higher than that of the WT-CLP group (p = 0.035); and PCPA had a protective effect on the WT+PCPA-CLP septic shock group, which had a higher survival rate than the WT-CLP septic shock group 3 days after the CLP (p = 0.048). However, there was no difference between the KO+5-HTP CLP septic shock group and the KO-CLP group (p = 0.086). The above results demonstrated that 5-HT deficiency provided a significant level of protection.The animals (ic shock . A log-rp < 0.05). Meanwhile, the body temperature of the mice in the KO-CLP sepsis group was higher than the WT-CLP sepsis group and was much closer to the normal body temperature after 24 hours (p < 0.01)  .p < 0.05) (p < 0.05) (p < 0.05) (p < 0.05) (p < 0.05)  . Moreove < 0.05) . In the  < 0.05) . In the  < 0.05) . In the  < 0.05) . In the  < 0.05) . Based oα and IL-6 (α and IL-6 secretion was observed in the CLP sepsis group compared with the sham group (p < 0.05). Moreover, a significant inhibition of TNF-α was discovered in the KO-CLP sepsis group in contrast to the WT-CLP sepsis group both at 6 h and 24 h after the CLP (p < 0.05), while the KO-CLP mice had a lower level of IL-6 at 24 h rather than at 6 h after the CLP compared with the WT-CLP mice (p < 0.01). In conclusion, 5-HT increased serum cytokine secretion, such as TNF-α and IL-6, and aggravated the mortality of sepsis by an excessive inflammatory response.The inflammatory response plays a fatal role in the process of sepsis. Serum cytokines were assessed by determining the serum levels of the TNF-and IL-6 . A markep < 0.05).Bacteria and their metabolic products are the initial cause of sepsis. The peritoneal fluid, blood, and lung tissues were collected for measuring the bacterial loads . We discp < 0.05)  . DHE flu < 0.05) .α and IL-6, and an excessive inflammatory response; (2) the acceleration of bacterial translocation; and (3) the promotion of oxidative stress, for instance, MPO and ROS. All the evidence illustrated that 5-HT could increase the risk of sepsis. In contrast, the5-HT deficiency provided a significant level of protection. Bacteria and their metabolic products caused an uncontrollable inflammatory response and played a crucial role in sepsis. The relationship between bacterial translocation and sepsis seemed to be a positive regulation. The previous study proves that there is a close association between bacterial translocation and gut-derived sepsis and bacterial translocation has been put forward as a concept to explain sepsis without an infectious focus [In this study, a model of sepsis and septic shock was established in mice by CLP and the influences of 5-HT on survival rate, sepsis manifestation, organ dysfunction, serum cytokines, bacterial loads, and oxidative stress were assessed to investigate whether 5-HT drives mortality and the potential mechanism in the process of sepsis. With this work, we demonstrated that 5-HT markedly increased mortality in sepsis and septic shock. The study further exhibited that 5-HT exacerbated the general manifestation as well as caused histological damages in the lung, liver, kidney, intestinal tissue, and heart. The mechanisms that 5-HT aggravated the sepsis might include the following: (1) the increasing secretion of serum cytokines, such as TNF-us focus , 34. Meaus focus –37. It hus focus . In our Our findings showed that 5-HT exacerbated the histological damages in the lung, liver, kidney, intestinal tissue, and heart, which resulted in organ dysfunction. A recent research claimed that a sepsis-induced elevation in plasma 5-HT facilitated the endothelial hyperpermeability . In thisα is released by macrophages in response to infection, stimulates the production of downstream cytokines, such IL-6 and IL-8, and plays a significant role in activating the cytokine cascade [α and IL-6 in the plasma were also substantially increased in CLP-induced sepsis [α were decreased in the 5-HT deficiency group at both 6 h and 24 h. However, the absence of 5-HT inhibited the level of IL-6 at 24 h but not at 6 h after the CLP. The possible reason why the KO-CLP mice and the WT-CLP mice showed no differences 6 h after the CLP administration might be that IL-6 was not massively released at the early stage of inflammation.TNF- cascade , 42. In d sepsis . Our stuThe effect of oxidative stress has been widely studied in previous studies , 44, 45.In this study, we discovered that 5-HT drove mortality and exacerbated organ dysfunction through the promotion of serum cytokines and bacterial loads and by facilitating oxidative stress in the process of sepsis. This study proved that 5-HT regulated sepsis progression and might be a new therapeutic target for sepsis. However, the mechanism of increasing bacterial loads induced by bacterial translocation has no direct evidence and further studies are required. Whether the mechanism of endothelial hyperpermeability and aggravating injury plays a key role in histological damages and organ dysfunctions induced by 5-HT needs further studies. Moreover, prospective clinical studies are needed to evaluate whether the level of 5-HT relates to the process of sepsis.In conclusion, the results of the present study demonstrated that 5-HT markedly drove mortality and exacerbated organ dysfunctions in sepsis. The potential mechanisms might be that 5-HT facilitated the inflammation, bacterial loads, and oxidative stress. These findings indicated that 5-HT played a key role in the process of sepsis and might be a potential therapeutic option in sepsis.


ACSL1 and ELOVL6 were critical genes in hepatic lipid metabolism and had opposite expression patterns with aan-miR-203a and aan-miR-125b-5p, respectively. And we validated that aan-miR-203a and aan-miR-125b-5p might involve in the regulation of hepatic lipid metabolism by targeting ACSL1 and ELOVL6, respectively. These results add to our current understanding of the regulation network in goose lipid metabolism.Unlike mammals, in palmipedes de novo lipogenesis from diet takes place mostly in the liver. The French Landes Goose is famous for its high capacity and susceptibility to fatty liver production. While miRNAs play a critical role in the posttranscriptional regulation of gene expression, miRNAs that are involved in the regulation of goose hepatic steatosis have yet to be elucidated. Using high-throughput sequencing, we analyzed miRNAs expression profile of Landes goose liver after overfeeding for 21 days. Aan-miR-122-5p was the most frequently sequenced known miRNA, but it was unchanged after overfeeding. Compared with normal liver, we identified that 16 conserved miRNAs were up-regulated while the other 9 conserved miRNAs were down-regulated in fatty livers. Many of their predicted target genes played key roles in metabolic pathways leading to the development of hepatic steatosis in the goose by KEGG pathways analysis.  In contrast, in wild palmipedes under natural conditions, hepatic steatosis occurs as a consequence of evolutionary adaption to accumulated reserves needed for migration2. In poultry production, this specific capacity is exploited for the production of “foiegras”3. Geese form fatty livers after feeding on a carbohydrate-rich diet for less than two weeks. And the liver weight may increase more than 10-fold and up to 10% of BW4. The French Landes Goose is famous for its high capacity and susceptibility to fatty liver production.The livers of animals are important tissues of lipid metabolism. In humans, hepatic steatosis is typically the causal factor for abnormal liver function and is frequently correlated with a variety of metabolic disorders, such as obesity, insulin resistance, and type II diabetes5. MicroRNAs (miRNAs) are 20–24-nt long, endogenous small noncoding RNAs. They can hybridize to sites in the mRNAs of protein-coding genes to direct posttranscriptional regulation of gene expression by inhibiting translation and/or inducing degradation of target mRNAs7. To date, evidences indicate that miRNAs are key trans-acting factors and a single miRNA can repress the production of hundreds of protein coding target genes through relatively mild means7. Dysregulated expression of miRNAs has been reported in human fatty liver production9. And many miRNAs have been well characterized for their roles in regulating lipid metabolism, such as miR-12210. Nevertheless, so far there is a scarcity of information available regarding the abnormal expression miRNAs and their target genes in fatty liver of goose. Thus, the studies on lipid metabolism-related miRNAs and their mechanisms in goose liver could serve as an important reference for poultry breeding and human liver disease research.In order to find the mechanism of the development of fatty liver, some researchers have studied the crucial genes involved in hepatic steatosis of waterfowlThe goal of this study was to identify differentially expressed miRNAs in the fatty liver of the Landes goose by using high-throughput technologies, deep-sequencing analysis, and qRT-PCR. Furthermore, we have predicted the target genes of the miRNAs and preliminarily validated some miRNA-mRNA pairs involved in the development of goose fatty liver. These findings provided insights into the role of miRNAs in the hepatic steatosis of goose.P < 0.01), respectively. The liver accounted for 3.92% of total body weight in the control geese and 10.17% in overfed geese. The increased liver weight accounted for 21.80% of total increase in body weight. By histological examination, the livers from control geese and overfed geese displayed marked differences. In Fig. As shown in Fig. By sequencing analysis, the distributions of all small RNA classes in the two groups were displayed in Table P < 0.05). Conversely, the levels of aan-miR-30d, aan-miR-125b-5p, and aan-miR-146a-5p in overfed groups were only 42.60%, 62.09%, and 54.08% of those in the control groups (P < 0.05), respectively. The fold-change was differential between high-throughput sequencing and qRT-PCR. However, the variation trend was consistent between the two detection methods.We confirmed 6 miRNAs using qRT-PCR Fig. . Aan-miRA. anser, we analyzed the putative target genes of differentially expressed miRNAs and classified the putative targets according to their pathways by KEGG pathway analyses. Figure ACSL1 and ELOVL6 transcripts may be the target genes of aan-miR-203a   Fig. . Comparemic Fig. . The intmic Fig. .Figure 411, such as nonalcoholic fatty liver disease (NAFLD)12, alcoholic fatty liver (AFL)13, etc. miRNAs have been reported to play important roles in the regulation of glucose and lipid metabolism in the liver15. Domestic Palmipedes are particularly susceptible to hepatic steatosis, and unlike human NAFLD, the fatty liver is nonpathological and reversible16. Goose liver has been widely studied by researchers as a good model to investigate the mechanism of lipid metabolism17. Since the miRNAs involved in fatty liver production in geese remain to be elucidated, an analysis of the functional miRNAs involved in goose hepatic steatosis was performed on.To date, many researchers have focused on liver diseases in mammalsA. anser genome database for matching analysis18 and the miRNAs profile of goose hypothalamus for reference19. It provides a more accurate platform for studying the roles of miRNAs in fatty liver production in geese. In this study, we found that 25 conserved miRNAs were differentially expressed in goose liver after overfeeding. In addition, we verified several miRNAs by qRT-PCR. Although some of the fold changes obtained by qRT-PCR were not identical to those obtained by deep sequencing, the trend in the changes was consistent across the two techniques.In our study, the size, color, weight and histology of the fatty liver showed that the livers of geese produced obvious hepatic steatosis after 21 days overfeeding. It provides a good animal model for fatty liver research. In addition, our lab possesses the 20. MIR221 was upregulated in adipose tissue of obesity individuals21. Similarly, the expression of aan-miR-222 and aan-miR-221 was increased as indicated by our study. MIR30d was a key regulator of human adipocyte development22. MIR148a controlled the expression of key proteins involved in cholesterol-lipoprotein trafficking23. Mir29 was downregulated in fatty livers of ob/ob mice24. Consistent with these studies, the expressions of aan-miR-30d, aan-miR-148a and aan-miR-29a significantly reduced in fatty liver of goose.Some dysregulated miRNAs in this study were consistent with known changes observed in other studies focused on lipid metabolism. MIR222 was upregulated in NASH and its predicted target genes are known to be involved in carbohydrate metabolism25. Mir122 knockdown in mice reduced the cholesterol levels in the circulation and liver, decreased fatty acid synthesis, and increased fatty acid oxidation in the liver26. Mir122 was downregulated in rats treated with high-fat diet and enriched with or without fructose27. However, in our study, the expression of aan-miR-122-5p showed no significant change. And this result is consistent with the change in duck with high fat diet28. MIR375 is involved in the regulation of insulin release and glucose homeostasis29. Inconsistent with the underexpression of MIR375 in NASH30, aan-miR-375 was significantly increased in the fatty liver of goose. It is worth noting that some miRNAs have different change in same processes in different species. For example, MIR125b was abnormal expression in GBM5 cell line treated with ARA and DHA but not in the other, although all the studied cell lines were glioblastoma cells31. These differences can be attributed to the presence of different signaling pathways and can be species-specific. Elucidation of the specific functions of these dysregulated miRNAs in the development of goose fatty liver requires further experimental data.Aan-miR-122-5p is a miRNA with tissue-specific expression and showed predominance in the liver. Consistent with mammals, it accounted for at least 70% in the two groups. Many studies have suggested that MIR122 is important for the regulation of hepatic lipid metabolism32 and alcoholic steatohepatitis33 in rat models. Mir125b has been shown related to adipocyte differentiation and adiposity35. However, few studies described their function in hepatic lipid metabolism. ACSL1 and ELOVL6 are predicted target genes of aan-miR-203a and aan-miR-125b-5p, respectively. ACSLs play a critical role in the first committed step in fatty acid metabolism by the thioesterification of long-chain fatty acids into their acyl-CoA derivatives36. ELOVL6 is responsible for the elongating C12-C16 to longer fatty acids37. In addition, ACSL1 and ELOVL6 had opposite expression patterns with aan-miR-203a and aan-miR-125b-5p, respectively. Relative luciferase activity values of recombined Dual-Luciferase reporter vectors with 3′-UTR of ACSL1 and ELOVL6 were significantly decreased by aan-miR-203a and aan-miR-125b-5p mimics, respectively. These results suggest that aan-miR-203a and aan-miR-125b-5p may implicate in the regulation of hepatic lipid metabolism by targeting ACSL1 and ELOVL6, respectively. But because anti-ELOVL6 and anti-ACSL1 antibodies specific for goose were lacking, the effect of miRNAs on the protein expression level of their target genes were not measured. However, the overexpression of aan-miR-203a and aan-miR-125b-5p in primary hepatocyte had no significant impact on lipid accumulation. Therefore, the specific function of aan-miR-203a and aan-miR-125b-5p on lipid metabolism of goose need further research.In order to understand the biological function of differential expression miRNAs, we predicted their target genes and classified these genes according to their pathways by KEGG pathway analyses. Many of these target genes play key roles in metabolic pathways leading to the development of hepatic steatosis in the goose fatty liver. Mir203a can be used as the potential biomarker for type 2 diabetesA. anser and identified some conserved miRNAs with significant differences in expression in the fatty liver that may play an important role in hepatic steatosis. These results add to our current understanding of the regulation network in goose lipid metabolism.Goose liver can be a good model to investigate the mechanism of lipid metabolism. In summary, we built fatty liver model of the All experimental protocols were approved by Animal Ethics Committees of Nanjing Agricultural University and Zhejiang Academy of Agricultural Sciences, China. The slaughter and sampling procedures complied with the “Guidelines on Ethical Treatment of Experimental Animals” (2006) No. 398 set by the Ministry of Science and Technology, China. All efforts were made to minimize animal suffering.The experiment was carried out using 90 days-old male Landes Geese hatched on the same day. 60 animals were divided into two groups and bred under natural conditions of light and temperature. All geese were housed in individual cages during the trial period. The carbohydrate-rich diet contains 3500 kcal, 4.8 g fat, and 100 g protein/kg. For 1–2 days of overfeeding, the animals were fed 150 g each time and 2 times per day. For 3–7 days of overfeeding, the animals were fed 150 g at a time and 3 times per day. At the end of overfeeding, they were fed 300 g at a time and 4 times per day. As control, geese were given a growing diet  and ad libitum during the trial period. Animals had free access to water at all times. Ten liver samples, including five control and five experimental geese (overfed for 21 d), were prepared for histological studies and analyzed for the expression of miRNAs. Prior to sampling, the geese underwent fasting for 12 h and only water was provided. The workflow can be found as Supplementary Figure The geese were overfed for 21 days and the control geese were sacrificed at the same time. The livers of 10 geese were collected and fixed in formalin solution for 24 h, and then embedded in paraffin and sectioned. The slices were stained with hematoxylin-eosin (HE) and observed by microscopy.19. High-throughput sequencing was performed with Illumina HiSeq. 2000 system. After removing the adaptors, low quality tags and contaminants reads, the clean reads were aligned with the goose genome (https://www.ncbi.nlm.nih.gov/nuccore/AOGC00000000). Then we filtered the known non-miRNA reads, such as rRNA, tRNA, snRNA, and snoRNA by screening against ncRNA deposited in the GenBank and Rfam databases. The remaining reads were searched against the miRbase 21 to identify the conserved miRNAs (http://www.miRbase.org/). The conserved miRNAs were named according to the guidelines for the miRNA terminology38. The expressions of miRNAs were normalized by Fragments per Kilobase of Transcript per Million Fragments Mapped (FPKM)39. Differentially expressed miRNAs were identified based on FPKM ≥ 100.00 in either of the two groups, fold change ≥ 1.50 or ≤ 0.67, and P < 0.001. Potential target genes of differential expressed miRNAs were predicted by matching miRNA-3′-UTR sequence and assessing their energy stability using Miranda algorithm40. And then GO Term (http://geneontology.org/) and KEGG pathway analyses (http://www.genome.jp/kegg/) of target genes were performed. The miRNA-mRNA interaction network was constructed using Cytoscape 3.5.1 software.Total RNA of each sample was extracted with Trizol . Using a Novex 15% TBE-Urea gel, 20–30-nt long RNA fragments were isolated and purified from 10 μg of total RNA. The small RNA molecules were prepared for high-throughput sequencing according the method described previously−△Ct.Total miRNAs from the livers were extracted using the miRcute miRNA isolation kit . miRNAs were reverse transcribed to cDNA with the miRcute miRNA First-Strand cDNA Synthesis Kit  and specific stem-loop primers. mRNAs were reverse transcribed to cDNA with PrimeScript RT reagent Kit with gDNA Eraser . Primers used in the qRT-PCR were listed in Supplementary Table ACSL1 and ELOVL6 were amplified and recombined to pmirGLO Dual-Luciferase miRNA Target expression Vector  according to the manufacturer’s introductions. The verified vector plasmid was co-transfected into goose primary hepatocytes with miRNA mimics or negative control  by lipofectamine 2000 . After 48 h of transfecting, the luciferase activities were measured with Dual-Luciferase Reporter Assay System .The 3′-UTRs of goose 41. Cells were cultured in RPMI1640 medium containing 10% FBS  and incubated at 5% CO2, and 37 °C on 24-well cell culture dishes  at a density of 3 × 105/cm2. Medium was changed every 24 h. Cells were distributed into 4 groups after 24 h growth: blank control group (BC), negative control group (NC), aan-miR-203a mimic group and aan-miR-125b-5p mimic group. Transfections were performed using Lipofectamine 2000  according to the manufacturer’s introductions. Cells were collected after 48 h of tranfection to detect the expression of ACSL1 and ELOVL6. The intracellular lipid contents of hepatocytes were monitored using multimode reader  after Nile Red staining.Goose hepatocytes were isolated from 1–2 week-old Landes goose following the perfusion methodt-test. Differences were considered significant when P < 0.05. All data are expressed as the mean ± standard deviation (S.D.).Differences between two groups were analyzed using the SPSS software based on two-tailed Student’s https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc = GSE99200). All data generated or analyzed during this study are included in this published article (and its Supplementary Information files).Our miRNAs dates of high-throughput sequencing are available in NCBI (GEO99200, supplementary information


Drought in South Asia affect food and water security and pose challenges for millions of people. For policy-making, planning, and management of water resources at sub-basin or administrative levels, high-resolution datasets of precipitation and air temperature are required in near-real time. We develop a high-resolution (0.05°) bias-corrected precipitation and temperature data that can be used to monitor near real-time drought conditions over South Asia. Moreover, the dataset can be used to monitor climatic extremes  in South Asia. A distribution mapping method was applied to correct bias in precipitation and air temperature, which performed well compared to the other bias correction method based on linear scaling. Bias-corrected precipitation and temperature data were used to estimate Standardized precipitation index (SPI) and Standardized Precipitation Evapotranspiration Index (SPEI) to assess the historical and current drought conditions in South Asia. We evaluated drought severity and extent against the satellite-based Normalized Difference Vegetation Index (NDVI) anomalies and satellite-driven Drought Severity Index (DSI) at 0.05°. The bias-corrected high-resolution data can effectively capture observed drought conditions as shown by the satellite-based drought estimates. High resolution near real-time dataset can provide valuable information for decision-making at district and sub-basin levels. The South Asian region faced multiple severe and long-lasting droughts, which posed tremendous impacts on growing economies of the region. India, Pakistan, and Sri Lanka have reported frequent droughts (once in every three years) in the last decades. In India, about 330 million people were affected due to the 2014–15 drought which had a return period of 542 years2 and led to a severe water shortage. Pakistan also experienced many droughts, and the 1999–2000 (continuing up to 2002) drought affected about 3.3 million people and resulted in a negative growth of 2.6% in the agriculture sector3. Due to severe droughts, the Pakistan government announced emergency conditions4. In Bangladesh, 19 droughts occurred between 1960 and 1991. On an average, a severe drought occurs in Bangladesh once in every 2.5 years5 and affects 53% of the population and 40% of crop production. Currently (in 2017), South India and Sri Lanka are facing a severe drought that has already affected more than 0.2 million people.Drought is one of the most complex natural disasters, which is often difficult to identify, , predict, and mitigate. In South Asia, increasing population and frequent droughts have been significant factors in furthering the water crisis and food scarcity. The drought of 2002 was among the most severe events, affecting around 300 million people in India7 . In South Asia, only India has a near real-time drought monitoring system, however, at a coarser spatial resolution (0.25°). The Indian experimental drought monitor system8 provides near real-time (1-day lag) drought information. The India Meteorological Department (IMD) (www.imdpune.gov.in) also provides drought information in India, however, at monthly scale and much coarser spatial resolution. The Pakistan Meteorological Department (PMD) (http://www.pmd.gov.pk/) provides monthly drought information using rain gauge station data and satellite-based vegetation index. The satellite-based near real-time drought monitoring and early warning system  for Asian Pacific countries9 provide drought warning at the state level. Drought monitoring systems at global scales10 have also been valuable for decision making at national levels.A high-resolution near real-time drought monitoring system is required for South Asia to assist policy makers and water managers and to minimize the detrimental impacts of water and food scarcity. Drought information at district to village level is needed for decision making and adaptation8–10 provide information at the regional and global scales, those are unable to help in decision making at the local scale (sub-basin or district levels) due to their coarse spatial resolution. A high-resolution near real-time precipitation and air temperature (maximum and minimum) dataset is required for drought monitoring at the district and sub-basin levels in South Asia. Here we use Climate Hazards Group of Infra-Red Precipitation with Stations (CHIRPS11) and maximum and minimum air temperatures from Global Ensemble Forecast System (GEFS) reforecast version 2 (ref. 13 (SPI) and Standardized Precipitation Evapotranspiration Index14 (SPEI) at 0.05° spatial resolution. The drought information at high resolution can be useful for policy makers, stakeholders, and water managers to in decision-making and as well as in climate change adaptation.While existing drought monitoring systemsn 2 ref.  from 19815, which are available at 0.25° spatial resolution over the Asian Monsoon region (V1101R1) for the period of 1981–2007. The APHRODITE data (V1101R1) were developed using station based observations, which represent spatial variability and other rainfall characteristics well. For instance, APHRODITE data (here onwards: APHRO-Precipitation) represent orographic rainfall in the foothill of the Himalaya and the Western Ghats. The data are well quality controlled and checked for inconsistencies and errors15. Recently, Xie et al.16 used APHRO-Precipitation to investigate droughts in Pakistan while Duncan et al.17 used the dataset to analyse temporal trends in the Indian summer monsoon.We obtained daily gridded precipitation data from Asian Precipitation Highly Resolved Observational Data Integration Towards Evaluation of Water Resources (APHRODITE)18, which are available at 0.25° and 0.05° spatial resolutions. CHIRPS-Precipitation is a combined product of monthly precipitation climatology (CHPClim), Thermal Infrared (TIR) satellite observations, and in situ precipitation observations from various national and regional meteorological departments18. The CHPClim is a long-term historical average rainfall accumulation, which is temporally disaggregated into 72 pentads (6-pentad per month) at a spatial resolution of 0.05°  precipitation data were obtained from the CHIRPS product (here onwards: CHIRPS-Precipitation)05° ref. . The The22, http://hydrology.princeton.edu/data/pgf/0.25deg/daily/). Princeton-Temperature was obtained for the period 1970 to 2007 at 0.25°, which was developed using global observed datasets from the National Centers for Environmental Prediction (NCEP) and National Center for Atmospheric Research (NCAR) reanalysis data22. Princeton-Temperature was compared with the observed India Meteorological Data (IMD) and it was found that both the datasets are consistent with spatial and temporal variability and for correlations between precipitation and air temperature23. Recently, Princeton-Temperature was used to evaluate the changes in hydro-climatic variables over the Indian subcontinental basins23. Moreover, Princeton-Temperature and precipitation datasets have been widely used to assess global and regional drought characteristics24–27.Since a consistent long-term daily temperatures (maximum and minimum) dataset is unavailable for the South Asia, we obtained gridded daily maximum and minimum temperature data from the University of Princeton (here onwards: Princeton-Temperature28 (https://www.esrl.noaa.gov/psd/forecasts/reforecast2/download.html) for the period of 1985 to present at 0.50°. GEFS-Temperature is generated on a daily basis at 0000UTC  which contains ten perturbed forecast members and one control forecast member. GEFS-Temperature was regridded at 0.25° using the synergraphic mapping system (SYMAP) algorithm. The SYMAP algorithm uses temperature lapse rate using a high-resolution elevation, which is discussed in detail in Maurer et al.29. GEFS-Temperature has been used as a forcing in land surface models to evaluate drought severity, monitoring, and forecast30–32. We used a high resolution  maximum and minimum temperature climatology from the Worldclim version 2 (here onwards: WCLIM-Temperature)34 (http://worldclim.org/version2) to provide spatial variability in the regridded GEFS-Temperature at 0.05°. The WCLIM-Temperature monthly climatology is available for the period of 1970–2000, which was interpolated using the thin-plate smoothing spline algorithm implemented in the ANUSPLIN package34.Since Princeton-Temperature is not available in near real-time, maximum and minimum 2-m air temperature data were obtained from the Global Ensemble Forecast System (GEFS) reforecast version 2 (here onwards: GEFS-Temperature)35. We evaluated bias in CHIRPS-Precipitation and GEFS-Temperature against APHRO-Precipitation and Princeton-Temperature, respectively. There are several approaches available for bias correction36–39; we analysed two bias correction methods  to adjust the bias in CHIRPS-Precipitation and GEFS-Temperature (minimum and maximum) and selected the most efficient method of bias correction that can reduce bias effectively in near real-time.Model analysis and satellite data may have random errors and bias compared to observations due to inadequate sampling, algorithm imperfection, and lack of in-situ data8; first, we applied the correction to extreme events  and then, bias correction was applied to total annual precipitation. We, therefore, estimated two scaling factors for each of 12 calendar months: i) for extreme values  and ii) for annual totals of precipitation for each grid cell. The monthly scaling factors for extreme events were determined by taking the ratio of the sum of the extreme precipitation data of APHRO-Precipitation and CHIRPS-Precipitation for the corresponding month. Then, pentad CHIRPS-Precipitation were corrected by multiplying the particular month’s scaling factors for each grid assuming that scaling factors remain constant for all six pentads within a month. After adjusting the extreme events of all months, we estimated monthly scaling factors for annual totals by taking the ratio of total precipitation of APHRO-Precipitation and CHIRPS-Precipitation for the corresponding month and corrected the pentad CHIRPS-Precipitation by multiplying the scaling factors.CHIRPS-Precipitation was bias-corrected using the linear scaling and distribution mapping methods. In the linear scaling, bias correction of CHIRPS-Precipitation was performed in two steps as described in Shah and Mishra41, bias correction was performed by matching the cumulative distribution functions (CDFs) of datasets where a particular distribution  was used to estimate the parameters. We used the Gamma distribution42 to fit pentad precipitation while empirical and normal distributions were used for temperature data.In the distribution mapping methodth using the To correct the precipitation data, we fitted the Gamma distribution to pentad precipitation to estimate the parameters for each month for APHRO-Precipitation and CHIRPS-Precipitation for each grid cell (0.25°). These monthly parameters for distributions were used for bias correction (to match the CDFS). We estimated the number of rainy pentads (>1 mm) in a given month for APHRO-Precipitation and CHIRPS-Precipitation for the training period. If CHIRPS-Precipitation has the number of wet pentads more than APHRO-Precipitation, we estimate the threshold value R29 to calculate the monthly scaling factors and parameters at 0.05°. We also evaluated the approach based on disaggregation of monthly scale factors and results were similar. These monthly parameters were used to correct CHIRPS-Precipitation at 0.05° using the same approach. The bias-corrected precipitation at 0.05° was aggregated to 0.25° and compared with the corrected CHIRPS-Precipitation at 0.25°, which was corrected with the APHRO-Precipitation at its native 0.25°. We found that both the bias corrected products resulted in a similar bias against the observed precipitation.The bias correction of pentad CHIRPS-Precipitation involves two steps. Initially, we corrected pentad precipitation at 0.25° using the linear scaling and distribution mapping. Then final correction of pentad CHIRPS-Precipitation was performed at 0.05° using the most efficient bias-correction method among the two. We corrected CHIRPS-Precipitation against APHRO-Precipitation at 0.25° for the training (1981–2004) period, and the effectiveness of bias correction was evaluated for the testing (2005–2007) period. We disaggregated the APHRO-Precipitation from 0.25° to 0.05° spatial resolutionWe also analysed the bias correction in 0.25° GEFS-Temperature (minimum and maximum temperature) against the observed Princeton-Temperature for training (1985–2004) and testing (2005–2007) periods using linear scaling method and distribution mapping (Q-Q mapping) method. In linear scaling method, we estimated monthly scale factors from both the temperature datasets for each month for each grid cell for the training period 1985–2004. The monthly scale factors were estimated by subtracting the mean monthly maximum or minimum GEFS-Temperature to Princeton-Temperature for the corresponding month. These monthly scale factors were subtracted from the raw pentad GEFS-Temperature for each month to obtain the corrected pentad GEFS-Temperature for each grid cell.41, we estimated empirical CDFs for each month for each grid from Princeton-Temperature and GEFS-Temperature for the training period. We corrected the GEFS-Temperature using CDFs mapping (Q-Q mapping)41 except extreme temperature events (less than 5 and more than 95% probability of exceedance). For extreme temperature events, normal distribution was employed. Further details on the linear scaling and distribution mapping method of bias correction in temperature data can be obtained from Shah and Mishra8 and Wood et al.41, respectively. Using the linear scaling and distribution mapping, we corrected the GEFS-Temperature for training and testing periods at the spatial resolution of 0.25° and selected the most efficient method for bias correction.In the distribution mapping methodet al.29. The regridded GEFS-Temperature data were again corrected using the WCLIM-Temperature climatology (1970–2000), which is available at 5 km using the linear scaling method. This correction was done to capture spatial variability of high-resolution WCLIM-Temperature data. Further details on the datasets used in this study are presented in The adjusted maximum and minimum temperatures were regridded at the spatial resolution of 0.05° from 0.25° for the period 1970-present using high-resolution digital elevation model (for lapse rate) and the SYMAP algorithm as described in Maurer, 43 to estimate potential evapotranspiration (PET), which is required for SPEI estimation. We used R SPEI package44 to determine SPEI using the log-logistic distribution. SPI and SPEI values indicate a wet condition for above zero and dry condition for below zero values. Normal (SPI/SPEI between −0.5 and 0.5), abnormal (SPI/SPEI between −0.5 and −0.8), moderate (SPI/SPEI between −0.8 and −1.2), severe (SPI/SPEI between −1.2 and −1.6), extreme (SPI/SPEI between −1.6 and −2.0), and exceptional (SPI/SPEI less than −2.0) category droughts were considered in our analysis45.After the bias correction of CHIRPS-Precipitation and GEFS-Temperature at 0.05°, we estimated SPI and SPEI for drought assessment and monitoring. SPI and SPEI are commonly used dimensionless drought indices, which are estimated by fitting a probability distribution. SPI is used to measure precipitation deficit or surplus while SPEI also considers the effect of air temperature over multiple time scales. We used the Gamma distribution to estimate SPI. We used Hargreaves method46 to evaluate the drought estimates from CHIRPS-Precipitation and GEFS-Temperature in South Asia. We obtained MOD13A3  NDVI from the United States Geological Survey (USGS) MODIS Reprojection Tool Web Interface (MRTweb) (https://mrtweb.cr.usgs.gov/) from January 2002 to June 2016. 1 km NDVI was aggregated to 0.05° using the majority resample tool in ArcGIS. This monthly product is generated using 16-days 1-km NDVI product by employing a weighted temporal average value (if data are cloud free) and the maximum value (in the case of clouds)48. The MODIS vegetation indices are being used in Global Monitoring of vegetation health, modeling hydrologic process, drought monitoring, and global and regional climate change impacts assessment47. NDVI is widely used in drought assessment, drought and vegetation health monitoring49–51. Recently, Asoka and Mishra52 used NDVI and hydro-climatic variables (soil moisture and sea surface temperature) to predict vegetation anomalies for India.We used Normalized Difference Vegetation Index (NDVI) anomalies and the Drought Severity Index (DSI)High-resolution pentad precipitation, maximum and minimum temperature, SPI and SPEI datasets (Data Citation 1) are available from Figshare for the South Asia domain, which includes India, Pakistan, Nepal, Bhutan, Bangladesh, and Sri-Lanka. High-resolution datasets are also aggregated at district and sub-basin levels and freely available online through an open repository from 1981 to 2016, which will be extended to near real-time. Details on data format and columns can be obtained from a readme file provided at the above link.53.We compared mean annual precipitation in CHIRPS-Precipitation against APHRO-Precipitation at 0.25° for the training period (1981–2004). We find that spatial variability of the APHRO-Precipitation is well represented in the CHIRPS-Precipitation . For ins36 compared to the linear scaling as it performs efficiently in near real-time applications. Therefore, we selected the distribution mapping over the linear scaling method for bias correction. We estimated bias at annual  against Princeton-Temperature at 0.25° using the linear scaling and distribution mapping methods . GEFS-Teperature , and sigperature . Howeverperature  and the et al.29. After regridding the GEFS-Temperature, the dataset was further corrected using the monthly climatology of WCLIM-Temperature to capture finer scale spatial variability (We regridded the bias-corrected (using the distribution mapping) 0.25° GEFS-Temperature to 0.05° using a high-resolution digital elevation map (to provide lapse rate) and SYMAP algorithm as described in Maurer iability . We compiability . We compiability . The finftp://ftp.chg.ucsb.edu/pub/org/chg/products/CHIRPS-2.0/diagnostics/stations-perMonth-byRegion/pngs/Southeast_Asia.station.count.CHIRPS-v2.0.png).We estimated SPI and areal extent (%) of drought using APHRO-Precipitation and bias corrected CHIRPS-Precipitation at 0.25° and 0.05° for the period of 1982–2007 over the South Asia . The areWe identified that the 1987 and 2002 were the two most severe drought years  during the entire period of 1981–2007. We compared drought severity and areal extents from CHIRPS-Precipitation (at 0.25° and 0.05°) and APHRO-Precipitation (at 0.25°) using 4-month SPI at the end of September, which represents accumulated precipitation during the monsoon season . Drought58, which is a ratio of the total number of drought events when both DSI and 4-month SPEI showed the drought (DSI<−0.6 and SPEI<−0.5) to the total number of drought events when only DSI showed drought for the period of 2000–2011  for four districts . These fDrought events have increased in South Asia during the recent decades. A high-resolution near real-time drought monitoring in South Asia can be used for decision-making and to redefine policies in the areas of water management and agriculture. We bias-corrected precipitation and temperature datasets from CHIRPS and GEFS at 0.25° and 0.05° using the distribution mapping. The primary requirement of real-time drought monitoring is to have long-term data extended till near real-time. The high-resolution dataset can be used for drought monitoring and assessment at the district and sub-basin level in near real-time. Moreover, the dataset can also be used to monitor and assess hydroclimatic anomalies in one of the most populated and agriculture intensive regions of the world. The bias corrected pentad precipitation from CHIRPS and maximum and minimum temperatures from GEFS are available at 0.05° from the 1981-till present. Using the high-resolution bias corrected data, drought indices (SPI/SPEI) were estimated and evaluated against satellite-based drought products. The validation provides the confidence that the dataset can capture areal extent and severity of drought at district and sub-basin levels in South Asia.59 in some regions due to the less number of gauge stations, which might have affected the bias-correction of the CHIRPS data. Despite these limitations, the performance of bias-corrected CHIRPS data was satisfactory at both 0.25 and 0.05 resolutions. Similarly, for air temperature, we assumed that GEFS-Temperature captures the effect of elevation (lapse rate) and spatial variability after disaggregation to the higher resolution. GEFS-Temperature was bias corrected using the Princeton and WCLIM-Temperature, which might also have bias due to sparse gauge stations in some regions of the South Asia. Overall, areal extent of droughts was monitored well using the bias-corrected CHIRPS-Precipitation and GEFS-Temperature. However, like any other gridded dataset, there may be limitations and uncertainties related to bias correction and interpolation methods. Random errors may be generated or modified during the bias correction and interpolation of the precipitation and temperature datasets60–63. The dataset can be improved further based on the increased availability of observational stations in future.While we have evaluated the high resolution dataset using satellite driven vegetation and drought indices, there are certain assumptions and limitations. For instance, we assumed that APHRO-Precipitation represents orographic and spatial variability after disaggregation from 0.25° to 0.05. Moreover, APHRO-Precipitation may have bias compared to other observed  dataHigh resolution (0.05°) datasets are aggregated at district and sub-basin levels and uploaded in Ascii (text) format from 1981 to present for every district and sub-basin in the South Asia. The data include precipitation, maximum and minimum temperatures for each pentad from 1981 to 2016. We also provide SPI/SPEI values for each district/sub-basin for each pentad considering 1–48 months duration to provide information on short to long-term droughts. The datasets can be easily used for drought analysis in any district and sub-basin in South Asia. Users can download data, and select country , state, district, and sub-basin. The analysis can be performed for retrospective and near real-time monitoring and assessment of hydroclimatic extremes . The dataset will be updated on a regular basis to make it near-real time. For gridded data (0.05 degree), interested users can directly contact to the corresponding author. More instructions on data format and details can be obtained from a readme file provided in the data link.How to cite this article: Aadhar, S. & Mishra, V. High-resolution near real-time drought monitoring in South Asia. Sci. Data4:170145 doi: 10.1038/sdata.2017.145 (2017).Publisher’s note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.


Alpha-Glutathione S-transferase (α-GST) is a liver enzyme which showed properties making it useful in assessment of liver cell damage. A number of studies demonstrated its early elevation in different hepatic insults, but its pattern in HCV was controversial. Consequently, we planned this work to study the significance of Serum Alpha-Glutathione S-Transferase (α-GST) assessment in hepatitis C patients with different alanine aminotransferase (ALT) patterns.Sixty-five untreated male patients with history of hepatitis C virus (HCV) positive antibodies and 21 healthy age- and sex-matched control subjects were enrolled in this study. Sera were collected for confirmation of the presence of HCV antibodies (by ELISA) as well as for assessment of the levels of α-GST, ALT, aspartate aminotransferase, alkaline phosphatase, gamma glutamyl-transferase, total proteins, albumin and HCV RNA. HCV RNA was detected by the reverse transcription polymerase chain reaction (RT-PCR). Based on ALT level, patients were divided into three groups. Twelve patients with normal ALT levels (NALT), 29 with near normal ALT levels (NNALT), and 24 with high ALT levels (HALT). All data were statistically analyzed for significance and correlation as well as sensitivity, specificity, positive and negative predictive values.The mean value of α-GST in HCV patients was significantly higher compared to the control with 82% sensitivity, 85% specificity, 98% positive predictive value and 63% negative predictive value. These results were more or less similar to the results of ALT and higher than the results of all the other assayed liver function tests. The sensitivity, and positive and negative predictive values of α-GST were lower than aminotransferases, but higher than the other assayed liver function tests in NNALT and HALT groups. Nevertheless, in NALT, these parameters were higher for α-GST than all the other assayed liver function tests including aminotransferases.Assay of α-GST has an adjuvant in evaluation of liver cell damage in HCV patients. However, its role is much more valuable in patients with normal aminotransferases for early detection of liver cell damage. Hepatitis C virus (HCV) is a critical factor of liver disease and one of the most important health issues worldwide . HCV infAlpha-Glutathione S-Transferase (α-GST) is an enzyme responsible for cellular detoxifying processes. Its wide hepatic distribution, high cytosolic concentration, and short plasma half-life make its monitoring more useful than conventional biochemical liver function tests as a marker of hepatocellular damage . ImmunohRegarding the HCV infection, controversial data were demonstrated. Namour et al showed low sensitivity of α-GST for cytolysis of hepatocytes , while FSixty-nine untreated male patients  with a history of seropositive HCV antibodies from the attendants of the outpatient clinic of the Suez Canal University Hospital were enrolled in this study. Twenty-one healthy male adults matched for age were considered as a control group. All had negative history of Hepatitis B and Bilharziasis. After obtaining written consents from all patients and controls, 5 ml of morning blood sample were collected in plain Vacutainer tubes to separate sera for the following: (1) Confirmation of the presence of HCV antibodies by the third generation enzyme-linked immunosorbent assay (ELISA) using the Abbott Murex anti-HCV (version 4.0) kit provided by Murex Biotec S.A. Ltd, Kyalami, Republic of South Africa. (2) Estimation of the serum level of alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP), gamma glutamyl-transferase (GGT), total proteins (TP), and albumin (Alb) by the automated Hitachi 912 using adaptations of the methods recommended by the International Federation of Clinical Chemistry. (3) Detection of HCV-RNA by the reverse transcription polymerase chain reaction where RT-PCR was done using TaqMan® One-Step RT-PCR Master Mix Reagents Kit , primers and a FAM labeled HCV probe for the 5′ non coding region of the HCV genome . An exogenous internal control  was included with each sample to assess the efficiency of the procedure. The lower limit of detection is 100 IU/ml. (4) Determination of α-GST by the ELISA HEPKIT provided by Biotrin International, Dublin, Ireland, according to the manufacturer’s instructions and as suggested by the manufacturer and the literature where α-GST levels ≤ 8 ng/ml were considered normal .t-test, correlation coefficient, and Chi-square test were used to evaluate the results. Chi-square test was used for qualitative variables, while independent t-test was used for quantitative variables. Correlation analysis was performed using Pearson's test. The sensitivity, specificity, positive and negative predictive values of each parameter were calculated according to the formulae described by Zhou et al [P-value < 0.05 and highly significance at P-value < 0.001.The data were coded and organized. The final study results were stated using the SPSS program version 14. Results were presented through Tables. Student’s ou et al . StatistELISA technique for assessment of HCV antibody levels showed results above the cut off value recommended by the manufacturer in sixty-five patient samples and below the cut off value in all control samples. The four patients with seronegative HCV antibodies were excluded from the study. Seropositive HCV patients (All HCV) were subdivided according to the suggestion of Giannini et al into three groups depending on the serum level of ALT . The firHepatitis C virus infection is usually monitored by ALT level. However, accumulating data indicate that ALT is not always correlated with liver cell damage, disease progression, or the response of HCV to therapy . MoreoveOn the other hand, GST system is indirectly involved in hepatocellular damage due to HCV . A signiThese data are not far from our results where we found a significant increase in the blood level of α-GST in all HCV group compared to the control group. Moreover, the sensitivity of α-GST was similar to the sensitivity of ALT (81.54%) in all HCV group and higher than the sensitivities of viremia, AST, ALP, GGT, TP, and Alb. In addition, the specifity of α-GST (84.88%) was not far from the specificity of ALT (86.05%) and higher than the specificities of viremia, AST, ALP, GGT, TP, and Alb. Although the positive predictive value of α-GST (98.15%) was lower than all the other assayed parameters, it was not far from the maximum. The negative predictive value of α-GST was more or less similar to ALT and higher than all the other assayed parameters. However, the positive predictive value of α-GST (95.24%) was slightly lower than all the other assayed parameters that were 100%.Correlation analysis showed significant positive correlation between α-GST and ALT, AST, ALP, and GGT which may indicate a common origin for these enzymes in such cases. A significant positive correlation was detected between α-GST and viral load which may be supported by the study of Giannini et al who showed significantly high HCV RNA load in patients with high α-GST .When we divided the HCV patients into three groups according to the level of ALT, the first group of patients with normal ALT (NALT) showed a significant increase in the α-GST level compared to the control (P < 0.02). Elevation of plasma level of α-GST may be an early indicator of liver damage. Different studies considered the plasma level of α-GST is an actual indicator of the integrity of the hepatocyte  or biomaThe sensitivity and specificity of α-GST in NALT group were higher than the sensitivity and specificity of ALT, AST, ALP, GGT, TP, and Alb. Except for viremia, the positive predictive value of α-GST was higher than all the other assayed parameters. The negative predictive value of α-GST was also higher than all the other assayed parameters including viremia. These data may indicate that α-GST may be more valuable than the other assayed parameters in evaluation of liver damage in patients with normal ALT, also, in cases where it is not possible to discriminate between "healthy" carriers and subjects with chronic liver damage, unless liver biopsy is performed. Nevertheless, the indication of liver biopsy in these conditions is not justified where authors considered it unethical to perform liver biopsies in patients with normal liver function tests . DetectiA significant increase of α-GST serum level was found also in NNALT compared to the control group. A significant positive correlation was found between α-GST and ALT, AST, and GGT within NNALT group that may support the relation of α-GST with the other liver function tests. The sensitivity of α-GST in this group (79.31%) was lower than the sensitivity of ALT which showed 100% sensitivity, but it was higher than the sensitivity of AST, ALP, GGT, TP, and Alb. Specificity of α-GST (86%) was found to be also lower than ALT (100%) but higher than the specificity of AST, ALP, GGT, TP, and Alb. Although α-GST showed lower positive predictive value (95.83%) than most of other investigated parameters, it had higher negative predictive value than all the other parameters except ALT. These data may indicate that assessment of α-GST is not superior to ALT in evaluation of liver damage in NNALT group. However, it may be related to viremia where a significant positive correlation is found between α-GST and viral load. Consequently, assay of α-GST in such patients may be used as an adjuvant with other liver function tests for evaluation of HCV infection.HALT patients showed also elevation of α-GST serum level which was significant compared to the control (P < 0.001). Comparing the sensitivity and specificity of α-GST with viremia of these patients, we found higher sensitivity and specificity of α-GST (91.67% and 93.33% respectively) than viremia (79.17% and 88.89% respectively). This may be attributed to the intermittent pattern of viremia -32. HoweIn conclusion, elevations of α-GST serum level in HCV patients with 81.54% sensitivity, and 84.88% specificity may give a role for its assessment in them. It may be assayed for confirmation of liver cell damage. However, its role would be much more valuable in early detection of liver damage in HCV patients with normal ALT where α-GST showed 66.67% sensitivity and 84.85% specificity compared to 0.0% sensitivity and 63.64% specificity for aminotransferases and other investigated liver function tests. It may be an indicator of liver biopsy in such conditions. The main limitation of the study is the relatively small sample size. Further research will be needed to confirm whether these results generalize to general population through a large community based study. In addition, further studies are needed to correlate any liver histopathological changes with serum levels of α-GST in cases of HCV with normal aminotransferases.


Fusarium graminearum species complex (FGSC), which produces trichothecene toxins. The FGSC is divided into three chemotypes: 3-acetyldeoxynivalenol (3-ADON), 15-acetyldeoxynivalenol (15-ADON), and nivalenol (NIV). In order to predict the geographical changes in the distribution of these chemotype populations in major winter wheat-producing areas in China, the biological characteristics of twenty randomly selected isolates from each of the three chemotypes were studied. No significant difference was exhibited in the growth rate of 3-ADON, 15-ADON, and NIV isolates at 15°C. At 20°C and 25°C, the growth rate of 15-ADON isolates was the highest. At 30°C, the growth rate of NIV and 3-ADON isolates was significantly higher than that of 15-ADON isolates. The 15-ADON isolates produced the highest quantities of perithecia and two to three days earlier than the other two populations at each temperature, and released more ascospores at 18°C. The aggressiveness test on wheat seedlings and ears indicated there was no significant difference between the 3-ADON and 15-ADON isolates. However, the aggressiveness of NIV isolates was significantly lower than that of the 3-ADON and 15-ADON isolates. The DON content in grains from heads inoculated with the 3-ADON isolates was higher than the content of 15-ADON and NIV isolates. The results showed that 15-ADON population had the advantage in perithecia formation and ascospore release, and the 3-ADON population produced more DON in wheat grains. We suggested that distribution of these three chemotype populations may be related to these biological characteristics.In China, Fusarium head blight is caused mainly by the  Fusarium graminearum species complex (FGSC). The FGSC can infect wheat during the period of wheat growth from seedling to ear formation stages, causing diseases such as seedling blight, crown rot, stem rot and ear rot ) for four hours. Samples were then centrifuged for five minutes at 4,000 rpm. Approximately 4 ml of the sample extract was gravity-filtered through Bond Elut Mycotoxin , a newly developed solid-phase extraction sorbent that cleans up food extracts for improved trichothecene analysis. A small portion (2 ml) of the clarifying solution was absorbed and transferred into a 15-ml centrifuge tube, and was evaporated with nitrogen at 50°C. The residue in the tube was dissolved in 0.5 ml of methanol-water (20:80 [vol/vol]). Then, this was further purified with a 0.22-μm organic microporous membrane filter, and the filtrate was transferred into a vial. The amount of DON was quantified using high performance liquid chromatography (HPLC)  .The method of trichothecene production in rice culture was modified according to the studies of Burlakoti et al.  and PuriData were analyzed using SAS software Version 9 . Conidium production and length, ascospore production, and trichothecene content were assessed using one-way analysis of variance (ANOVA). Mean mycelial growth rate was assessed at different temperatures using two-factor ANOVA analysis of variance. Because the data on aggressiveness were ordinal and not normally distributed, they were analyzed using the nonparametric method described by Shah et al. . PROC RAP = 0.9872), and there was an interaction between the chemotype and the temperature in the two-factor ANOVA analysis of variance. However, the mycelial growth rate of three chemotypes at some temperatures was singificantly different when the temperature factor was not taken into account. At 25°C, the growth rate of 15-ADON isolates was the highest. At 30°C, the growth rate of NIV and 3-ADON isolates was significantly higher than that of 15-ADON isolates, and the growth rate of NIV isolates was the highest  and NIV isolates (2.70 × 105 ascospore/ml)  .5 macroconidia/ml. The potential for the 3-ADON and NIV isolates to produce conidia were similar. Furthermore, the 15-ADON isolates had the longest macroconidia, which was significantly longer than those generated by the 3-ADON and the NIV isolates. However, there was no significant difference in macroconidia production between the 3-ADON and 15-ADON isolates  until O’Donnell et al. [F. graminearum. Since then, some reports have showed that F. graminearum of China falls into F. asiaticum and F. graminearum sensu stricto. F. asiaticum was the dominated species in the middle and lower reaches of the Yangtze River and F. graminearum is more common in northern China. 3-ADON and NIV were the main chemotypes in F. asiaticum. In F. graminearum, only 15-ADON chemotype was found [The primary pathogen of FHB in China was identified as l et al.  used genas found – 34. Basas found  found thF. asiaticum. NIV population appears more adapt to high temperature. The growth rate of the NIV population was lower at 15°C and significantly higher than that of 3-ADON and 15-ADON populations at 30°C. Moreover, in recent years, we have recovered stable proportions of NIV producing isolates [Zhang et al.  also revisolates , 35, butIn this study, we unexpectedly found that more perithecia were formed at an earlier period by the 15-ADON chemotype isolates. The 15-ADON chemotype population also released more ascospores than the other two chemotype populations. Spolti et al.  comparedIt has been well-accepted that the 15-ADON chemotype isolates predominate the FHB pathogen population in northern China, and the 3-ADON and NIV isolates are found mainly in the south. Based on results of this study, the advantage on perithecia formation and ascospore release may be critical for the 15-ADON population in the north, because the conditions for perithecia formation there such as temperature and moisture on the soil surface are less favorable than that in the south. In a previous study, 3-ADON isolates were found in the crown rot pathogen population even in the far north . This fiAlthough the capability of the 3-ADON population for perithecia formation and ascospore release was lower than that of the 15-ADON population, its aggressiveness and DON production in wheat were higher. With changes in global climate change and increasing temperatures, the conditions for perithecia formation are improving. Whether the 3-ADON population will move towards the northern regions of China and replace the 15-ADON population is worth monitoring.S1 FigF. graminearum species complex isolates.PCR amplification of chemotypes of 15-ADON (583 bp), 3-ADON (644 bp) and NIV (859 bp). M: 100 bp DNA ladder, Lanes 1–17 were (TIF)Click here for additional data file.S1 Table(DOCX)Click here for additional data file.


Caenorhabditis elegans to investigate the aversive behavior of nematodes to graphene oxide (GO) and the underlying neuronal basis.So far, how the animals evade the environmental nanomaterials is still largely unclear. In this study, we employed in vivo assay system of nlg-1 encoding a neuroligin with the function in connecting pre- and post-synaptic neurons suppressed the aversive behavior of nematodes to GO. Moreover, based on the neuron-specific activity assay, we found that the NLG-1 activity in AIY or AIB interneurons was required for the regulation of aversive behavior to GO. The neuron-specific activities of NLG-1 in AIY or AIB interneurons were also required for the regulation of GO toxicity.In this assay model, we detected the significant aversive behavior of nematodes to GO at concentrations more than 50 mg/L. Loss-of-function mutation of nlg-1 mutant as a genetic tool, we identified the AIY and AIB interneurons required for the regulation of aversive behavior to GO. Our results provide an important neuronal basis for the aversive response of animals to environmental nanomaterials.Using The online version of this article (10.1186/s12951-018-0373-y) contains supplementary material, which is available to authorized users. With the rapid increase in nanotechnology, a larger amount of engineered nanomaterials (ENMs) have been generated for industrial and medical applications. Among these ENMs, graphene nanomaterials have attracted massive attention due to their unique mechanical, electronic, and thermal properties , 2. GrapCaenorhabditis elegans has already been widely used in the toxicological study . A. AC. eledes Fig. b. In condes Fig. b. Therefnlg-1 in AIA interneurons or AIZ interneurons could not obviously affect the GO toxicity in inducing intestinal ROS production and in decreasing locomotion behavior in nlg-1(ok259) mutant nematodes  mutant nematodes , Al2O3-NPs (60 nm), multi-walled carbon nanotubes (MWCNTs), or thiolated GO (GO-SH) in nematodes (Additional file 2-NPs, Al2O3-NPs, MWCNTs, or GO-SH is available in the references [oxicants , 12. In ferences –43. ThesC. elegans, it has been shown that NLG-1/neuroligin is required for the control of synaptic function, a subset of sensory behaviors and sensory processing, longevity, and oxidative stress or stress response [nlg-1 significantly suppress the aversive behavior to GO (Fig. In response , 44–47.  GO Fig. , implyinnlg-1 in AIY interneurons or AIB interneurons could recover the deficits in aversive behavior to GO in nlg-1 mutant nematodes (Fig. nlg-1 in AIA or AIZ interneurons was not required for the function of NLG-1 in the regulation of aversive behavior to GO (Fig. We further provide the evidence to demonstrate the crucial function of interneurons in the regulation of aversive behavior to GO. Among the main classes of interneurons with the function to integrate sensory neurons with motor neurons , we obsedes Fig. . In cont GO Fig. . These r GO Fig. . BecauseC. elegans, genetic or laser ablation of AIY interneurons caused the abnormal spontaneous reversal rate, odorant chemotaxis, and salt chemotaxis [C. elegans, it was repotted that genetic or laser ablation of AIY interneurons can enhance the spontaneous reversal rate [In emotaxis –50. Addiemotaxis , 50. Oursal rate . Therefonlg-1 in the AIY interneurons in the regulation of GO toxicity [nlg-1 in the AIB interneurons in the regulation of GO toxicity (Fig. C. elegans, AIY interneurons act as the output of ASE, AWC, AFD, and AWA sensory neurons, and AIB interneurons act as the output of ASE, AWC, ASI, ASH, ASK, ADL, AFD, and ASG sensory neurons [C. elegans, the neurexin gene is nrx-1. The further examination of neuron-specific activities of NRX-1 will be helpful for final identification of neuronal circuit required for the control of aversive behavior to GO in nematodes.Our previous study has identified the neuron-specific activity of toxicity . In thisity Fig. . These rity Fig. . In C. e neurons . In C. eC. elegans. We observed the obvious aversive behavior of nematodes to GO at concentrations more than 50 mg/L. In nematodes, mutation of nlg-1 encoding a neuroligin disrupted this aversive behavior to GO. Using nlg-1 mutant as a genetic tool, we identified the AIY and AIB interneurons to be required for the regulation of aversive behavior to GO based on a series of rescue assays. Our results provide the important neuronal and molecular basis for the aversive response of animals to GO. Moreover, we found that both the AIY interneurons and the AIB interneurons were also required for the regulation of GO toxicity in nematodes.In this study, we investigated the aversive response of animals to GO using the in vivo assay system of Additional file 1. Additional table and figure.


Multicellular spheroids are very attractive models in oncology because they mimic the 3D organization of the tumour cells with their microenvironment. We show here using 3 different cell types , that when the cells are growing as spheroids the frequency of binucleated cells is augmented as occurs in some human tumours.We therefore describe mitosis in multicellular spheroids by following mitotic markers and by time-lapse experiments. Chromosomes alignment appears to be correct on the metaphasic plate and the passenger complex is well localized on centromere. Moreover aurora kinases are fully active and histone H3 is phosphorylated on Ser 10. Consequently, the mitotic spindle checkpoint is satisfied and, anaphase proceeds as illustrated by the transfer of survivin on the spindle and by the segregation of the two lots of chromosomes. However, the segregation plane is not well defined and oscillations of the dividing cells are observed. Finally, cytokinesis fails and the absence of separation of the two daughter cells gives rise to binucleated cells.Division orientation is specified during interphase and persists throughout mitosis. Our data indicate that the cancer cells, in multicellular spheroids, lose their ability to regulate their orientation, a feature commonly encountered in tumours.Moreover, multicellular spheroid expansion is still sensitive to mitotic drugs as pactlitaxel and aurora kinase inhibitors. The spheroids thus represent a highly relevant model for studying drug efficiency in tumours. For several decades, two-dimensional- 2D)-cell cultures were used for the screening of anti-cancer drugs, however they do not fully recapitulate the solid tumour architecture and its biology. Cells grown as monolayer, on 2D surfaces, present few cell-cell and cell–matrix interactions with a stretched and abnormal cytoskeleton rearrangement that lead to aberrant gene and protein expression. Additionally, the cells are uniformly oxygenated with a full access to nutriments. These changes result in accelerated doubling times. Three-dimensional 3D)-cultures emerged that realistically recapitulated tumour cell behaviour and have become, therefore, increasingly popular, in recent years. The architecture of 3D-cultures reflects the microenvironment of solid tumour with appropriated cell-cell interactions as well as gradients of nutrient and oxygen . 3D-multD-cell cuD-cultureSeveral systems of 3D-cell culture have been developed . In the These 3D systems are valuable tools for anti-cancer drug testing . CompariIn growing spheroids, the proliferating cells are preferentially located in the outermost layers . PreviouIn the present study, we report the presence of bi-nucleated cells in multicellular spheroids and we describe mitosis progression. The cell cycle analysis of 7 days old-spheroids indicated a decreased S-phase and a stable 4N-population that represents mitosis-arrested and tetraploid cells.We set-up time-lapse experiments on live fluorescent spheroids to follow mitosis. Direct live imaging has been poorly explored on spheroids. Our study demonstrates that the mitotic spindle checkpoint is satisfied since chromosomes are aligned on the metaphasic plate, histone H3 is phosphorylated on Ser10 and the passenger complex is transferred on the spindle. Conversely, cytokinesis failed as a consequence of the mis-orientation of the dividing axis, giving rise to binucleated cells.Despite unsuccessful mitosis, multicellular spheroids are a suitable model for testing mitotic drugs because their expansion is sensitive to pactlitaxel and aurora kinase inhibitors. Spheroids, thus, represent a highly relevant model for studying drug efficiency.Murine, p53WT TSA/pc cells derived from a mammary gland tumour were used for 3D-MCTS production either in U-low binding 96-wells or by the pending drop method. In both cases we identified a significant proportion of bi-nucleated cells within the spheroids Figure  and HeLaSince the presence of bi-nucleated cells is a consequence of unsuccessful mitosis, we studied the localization of important mitotic proteins and markers Figure . We perfAt the spheroid periphery, a cell in anaphase identified by its shape was continuously imaged Figure . This tiIn order to better describe the mitosis, we performed time-lapse experiments on HeLa cells expressing survivin-GFP. Survivin is a passenger protein localized on the centromeres as recalled in Figure The transfer of survivin from chromatin to microtubules indicates that the spindle checkpoint is satisfied when the cells are growing as spheroids Figure . As expeTo better define these unexpected features, we performed a time-lapse on cells expressing histone H2A-GFP Figure . We coulTo ascertain that cells escape from mitosis, experiments were performed with Fucci-HeLa cells expressing the two sensors: geminin-GFP and cdt1-RFP . GemininOne possible explanation for the mis-orientation of the cytokinesis axis could be a lack of tension between the daughter cells. We looked for the organization of fibronectin, an extracellular matrix protein. TSA/pc spheroids were expanded continuously Figure . When miThe analysis of the cycle revealed a decrease of S-phase; this phase represented 9 %, on day 4 and 4 % on day 7, for cells grown as spheroid Figure . The supIn adherent cells growing in 2D, P53 was described to be stabilized in bi-nucleated cells and to prevent the proliferation of tetraploid cells . Since tSpheroids are proposed as models for testing anti-cancer drugs since they better mimic tumours than adherent cells. Taking into account cytokinesis failure, we questioned whether the system was still suitable for testing mitotic drugs. We selected three anti-mitotic compounds: paclitaxel that prevents tubulin depolymerisation , AZD1172As a conclusion, we observed cytokinesis failure, in different cell-spheroids, as a consequence of the destabilization and mis-orientation of the segregation axis.in vivo tumours . TS. TS42]. Paclitaxel and AZD-1172 were supplied by Sigma-Aldrich. C5M was described in Le et al .2. Spheroids grew in complete medium as in 2D-cultures in the final volume of 200 μL.Spheroids were generated by plating either TS/A-pc or HeLa or Hek293 or Hct116 cells, at 1000, 800, 500 and 1000 cells/well respectively, into ultra low adherence −96-well plates . These plates stimulate spontaneous formation of a single spheroid of cells, within 24 hours of incubation at 37°C, 5% COMixed spheroids containing both TSA/pc tumour cells and NIH-3T3 fibroblasts were essentially prepared as already described in  except tAlternatively spheroids were prepared by the hanging drop technic. Drops of 40 μL  were pipetted on the lid of a petri dish containing PBS. The culture was maintained for 5 to 7 days.Spheroids were imaged in live conditions either directly in the 96-well plate or upon their transfer into coverglass Lab-Tek wells.For measuring their expansion, spheroids were imaged with a Zeiss510-confocal microscope, each day, with a 10-X objective and then the area of the larger section was determined for each spheroid by the Image J software. Most experiments were done on 5 spheroids alternatively on 3. Finally, at the end-point, the number of live cells was determined by the celltiter Glo-3D kit under conditions suggested by the manufacturer (Promega).For determination of cell cycle profiles, adherent cells were recovered and fixed by ice-cold 70% ethanol, for 1 hour, and then, incubated with propidium iodide solution (50 μg/ml) in the presence of 0.2 mg/ml RNase A for 15 minutes, at 37°C. DNA content was measured using the BD Accuri C6 flow cytometer  and CFlow Plus software.Determination of the cell cycle of cells grown as spheroids proceeded as follows: Five spheroids were recovered by pipetting in an eppendorf tube, washed by PBS then, transferred in a well of a 24-plate containing trysin/EDTA, incubated at 37°C with vigorously shaking from time to time. The dislocation of the spheroids is followed under the microscope and the action of trypsin stopped by adding FCS. Then, cells were recovered and cell cycle analysis is performed as described above.For 2D cultures, cell proliferation assays were conducted in 96-well culture plates. Assays were run in triplicate. Serial dilutions of compounds were prepared and the viable cell number was determined, on day 4, by the addition of MTS (Promega). Two different experiments were run. Dilutions are as follows: from 1 nM to 200 nM for AZD1152 and paclitaxel and from 100 nM to 1 μM for C5M.For 3D cultures, the growth was determined by measuring the maximal area of each spheroid and the percentage of expansion at Dx equals (area Dx – area D0/area D0) X 100. IC50 is the concentration that decreases expansion by 2. At the end-point, the number of live cells was determined by the celltiter Glo-3D kit (Promega). IC50 values indicated in Table Adherent cells grown on glass coverslips were fixed by formaldehyde 4%, for 10 minutes, at 37°C. Immunofluorescence staining was performed as described previously . ImmunofThe antibodies were targeted against the following antigens: α-tubulin , phospho-histone H3-Serine10 ; phospho aurora kinase-A-B-C , 1:100); phospho-AMPK , phospho-myosin light chain 2 , mouse P53 (CST 1: 100), Ki-67 , Cyclin B  and fibronectin (Sigma 1:500). Actin was stained by either phalloidin-rhodamin (1 μg/ml) or phalloidin-Atto 488 or phalloidin-Atto 647. DNA was visualized with either hoechst 33342  or NucRed . Images were collected with a ZEISS 710 Laser Scanning Confocal microscope with either a Plan-Apochromat 20×/0.75 or a water immersion 40 X objectives. Slices of 0.5 μm are shown as well as 3D-projections created by the zen software from Zeiss. A median filter (3/3/1) was applied to most images.For estimating the percentage of polyploid cells, DNA and polymerized actin were labelled. Then, photos were acquired with a confocal microscope and the scoring was manual. Around 1 000 cells were analysed. For 3D-cultures, the quantification was performed on z-stack images.Ex vivo experiments were conducted on cells grown on Lab-Tek chambered coverglass  and maintained under standard culture conditions . Spheroids were recovered by pipetting just before imaging and deposited in the Lab-Tek well containing cell culture medium. Images were acquired on a Zeiss dynascope confocal microscope using a Plan-Apochromat 20X / 0.75 objective. Images were analysed with the Zen software provided by Zeiss and a median filter (3/3/1) was applied to all images. At least 10 mitotic cells were simultaneously followed and three independent experiments were conducted.


Navigated transcranial magnetic stimulation (nTMS) has recently been introduced as a non-invasive tool for functional mapping of cortical language areas prior to surgery. It correlates well with intraoperative neurophysiological monitoring (IONM) findings, allowing defining the best surgical strategy to preserve cortical language areas during surgery for language-eloquent tumors. Nevertheless, nTMS allows only for cortical mapping and postoperative language deficits are often caused by injury to subcortical language pathways. Nowadays, the only way to preoperatively visualize language subcortical white matter tracts consists in DTI fiber tracking (DTI-FT). However, standard DTI-FT is based on anatomical landmarks that vary interindividually and can be obscured by the presence of the tumor itself. It has been demonstrated that combining nTMS with DTI-FT allows for a more reliable visualization of the motor pathway in brain tumor patients. Nevertheless, no description about such a combination has been reported for the language network. The aim of the present study is to describe and assess the feasibility and reliability of using cortical seeding areas defined by error type-specific nTMS language mapping (nTMS-positive spots) to perform DTI-FT in patients affected by language-eloquent brain tumors. We describe a novel technique for a nTMS-based DTI-FT to visualize the complex cortico-subcortical connections of the language network. We analyzed quantitative findings, such as fractional anisotropy values and ratios, and the number of visualized connections of nTMS-positive spots with subcortical pathways, and we compared them with results obtained by using the standard DTI-FT technique. We also analyzed the functional concordance between connected cortical nTMS-positive spots and subcortical pathways, and the likelihood of connection for nTMS-positive vs. nTMS-negative cortical spots. We demonstrated, that the nTMS-based approach, especially what we call the “single-spot” strategy, is able to provide a reliable and more detailed reconstruction of the complex cortico-subcortical language network as compared to the standard DTI-FT. We believe this technique represents a beneficial new strategy for customized preoperative planning in patients affected by tumors in presumed language eloquent location, providing anatomo-functional information to plan language-preserving surgery. Language is the result of the meaningful integration of complex parallel and sequential neuronal processing. The traditional view based on the assumption that Broca's and Wernicke's areas were the unique cortical areas, respectively, involved in the control of articulatory and semantic aspect of speech has been widely overpassed and replaced by a more complex interpretation based on the demonstration of a complex cortico-subcortical neuronal network plastically connecting different brain areas in the dominant perisylvian region, and sometimes also in the non-dominant hemisphere  existence of a brain tumor compressing/infiltrating areas presumably controlling speech production and comprehension and (2) obscured anatomy of language cortical perisylvian areas and/or language subcortical white matter tracts  due to the mass effect of the tumor or infiltrating growth. The exclusion criteria were: (1) frequent seizures (more than 1/week), (2) existence of a pacemaker or “deep brain stimulation” electrodes and (3) a clinical condition of severe aphasia hampering the ability to correctly perform the object-naming task needed for nTMS mapping. Preoperative aphasia grading of all patients was assessed and adapted to the Aachener Aphasia Test . The T1-weighted sequences  were usually used as reference anatomical exam. In case of non-contrast enhancing lesions , T2-weighted (TR/TE 5200 ms/100 ms) fast spin-echo sequences, or T2-weighted inversion recovery (TR/TE/TI 6000 ms/ 150 ms/ 2000 ms) fast spin echo sequence were used.All patients underwent preoperative 3 Tesla MRI scans for acquisition of T1-weighted 3D MPR, T2-weighted, FLAIR, and DTI sequences  with particular regard to the peritumoral area and, in general, to the perisylvian region of the dominant hemisphere. Cranial landmarks used for coregistration between the patient's scalp and MRI scan were the crus of the elix bilaterally, the nasion, and 9 more scalp regions identified by the nTMS software. The maximum stereotactic error allowed by the coregistration software was 5 mm. The stimulation procedure was blocked in case of a higher stereotactic error. Stimulation technique and parameters were already previously described  and the mean FA ratio  required for the best visualization of each fascicle by using the standard vs. the nTMS approach in general .The workflow for DTI-FT of language fascicles was performed by using the server-based planning software iPLAN NET 3.0  through a deterministic algorithm. DTI sequences were co-registered with the reference anatomical exam and with the DICOM images containing the nTMS mapping result. Coregistration consisted in a fully automated process performed by the neuronavigation software in order to increase the accuracy of image fusion. After correction for eddy currents, the software computed the FA, Apparent Diffusion Coefficient (ADC) and Directionally Encoded Color (DEC) maps. The DEC map was used for choosing the seeding ROIs for DTI-FT. Tracking was performed by using three different approaches: a standard atlas-based, a nTMS-based “all-spots”, and a nTMS-based “single-spot” strategy.Fiber tracking of AF, ILF, IFOF and UF was performed by placing ROIs over well-established anatomical landmarks as described in the literature  computed by using the standard approach were individually converted into a 3D object and used as first seeding ROI for the nTMS-based tracking. All nTMS spots were then segmented and selected all together as second ROI for fiber tracking . Again the FA value to stop tracking was set in order to obtain the best visualization of each fascicle, trying to reduce as much as possible the visualization of aberrant or artificial fibers. Once the best FA value was defined, the FA threshold and the FA ratio were calculated as described for the standard atlas-based strategy.As already described for the “all-spots” strategy, the 3D object of each fascicle obtained by using the atlas-based approach was used as first seeding ROI. Then, each single nTMS spot was segmented and used individually as second ROI. We repeated the computation for each single nTMS spot, each time using a different spot as second ROI and for all four main fascicles separately. The FA stop value was the same as the one used for the “all-spots” strategy for each of the main fascicles. This strategy was applied to avoid operator-related FA stop values selection bias that could alter the successive comparison of the number of connected spots obtained by using the two nTMS-based techniques. Finally, to further verify our approach, we repeated the same procedure by using three nTMS-negative spots individually selected as second seeding ROI in each patient, to test the assumption that nTMS-negative spots do not, or significantly less often, connect to main language fascicles.We analyzed the ability of the standard atlas-based strategy and of the nTMS-based approach to reliably compute language pathways without including aberrant or artificial fibers by comparing the mean FA stop values and ratios used for each fascicle. Then, we assessed the ability of the three techniques to visualize the complex cortico-subcortical language network by measuring and comparing the number of cortical nTMS-positive spots connected to the subcortical tracts. These connections were considered as true-positive. Each single connection visualized by the nTMS-based DTI-FT was considered not reliable if fibers (1) had a course that was completely different from the normal course of the corresponding fascicle according to its normal anatomy; (2) had a completely different course as compared to the normal anatomy of the corresponding fascicle, that could not be explained by a compression/dislocation/distortion caused by the tumor.In order to provide a preliminary statistical validation of our true-positive connections, we analyzed the eventual concordance between the presumed function of each fascicle and the prevalent error type of connected positive nTMS spots. Lastly, with the aim to assess the reliability of the DTI-FT reconstruction of the language network, we analyzed the number of connected nTMS-negative spots. These were considered as false-positive connections. According to the potential connection of each single spot (regardless positive or negative) to a maximum of 4 fascicles , we analyzed and compared the likelihood of false-positive subcortical connections with the likelihood of true-positive ones.t-test was used to compare (1) FA values and ratios, (2) the number of cortical nTMS-spots (positive or negative) connected to subcortical tracts obtained by using the three different techniques, (3) the likelihood of connections for positive and negative nTMS spots . The ANOVA test was used to analyze the different distribution of error types among connected nTMS-positive spots. Statistical significance was defined as a p < 0.05. GraphPad Prism version 6.00 for Windows was used for all the statistical analysis .Paired Student's We collected neuroimaging data of 10 patients  suffering from left perisylvian brain tumors  that underwent preoperative language mapping using repetitive nTMS. Table All patients underwent nTMS-mapping of the left perisylvian region in order to identify essential language sites Figure . We obseMean FA values and ratios were higher when using the nTMS-based approach  as compared to the standard atlas-based technique for each fascicle.The mean FA stop value was, respectively, 0.22 ± 0.03 vs. 0.19 ± 0.06 for AF, 0.20 ± 0.04 vs. 0.19 ± 0.06 for ILF, 0.17 ± 0.03 vs. 0.15 ± 0.04 for IFOF, and 0.15 ± 0.03 vs. 0.14 ± 0.04 for UF Figure . These dp = 0.006, p = 0.002, p = 0.04)  was 67.5 ± 7.2 vs. 55.8 ± 12.8 for AF, 85 ± 10.6 vs. 67.4 ± 10.6 for ILF, 76 ± 15.8 vs. 72.5 ± 10.6 for IFOF, and 77.6 ± 13.6 vs. 66.2 ± 18.1 for UF. The difference was statistically significant for AF, ILF and UF  Figure . In partp = 0.002; standard vs. nTMS “single-spot” p = 0.0004; nTMS “all-spots” vs. nTMS “single-spot” p = 0.009). Figure The total number of cortical nTMS spots connected to any of the language fascicles was significantly higher by using the nTMS-based “single-spot” strategy as compared to the other two techniques  by using the nTMS-based strategy for all fascicles Table .In particular, the nTMS-based “single-spot” strategy resulted in a significantly higher number of connected spots as compared to the other two techniques for AF, UF, and IFOF Figure .We report some examples of graphical differences between the nTMS-based and the standard atlas-based techniques in selected cases for AF Figure , UF Fig, and colp = 0.01), IFOF (p = 0.007), and ILF (p = 0.001). The distribution was not significantly different for the UF  vs. 10.9 ± 0.9 ; ] Figure .According to potential connections of both positive and negative nTMS spots, in our series we observed a likelihood of connections for nTMS-positive spots (true-positive connections) of 39.9 ± 6.7 %  vs. a likelihood of connection for nTMS-negative spots  of 9.1 ± 8.2 %  Figure .DTI-FT represents the only preoperative technique for visualization of the complex subcortical language network. Different studies have already demonstrated the utility of intraoperative neurophysiological mapping of subcortical language fascicles, especially if guided by DTI-FT, in reducing postoperative speech deficits after surgery of brain tumors located in the dominant perisylvian region , grant no. EXC1027/1, in conjunction with this study.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.